Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C2220/00—Biochemical treatment
  • A23C2220/20—Treatment with microorganisms
  • A23C2220/202—Genetic engineering of microorganisms used in dairy technology
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
  • A23V2400/123—Bulgaricus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
  • A23V2400/137—Delbrueckii
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/21—Streptococcus, lactococcus
  • A23V2400/249—Thermophilus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to mutants of lactic acid bacteria, such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, which are resistant to the antibiotic ampicillin, and which were found to give an increased texture when grown in milk while maintaining the other growth properties of the parent strain.
• the present invention furthermore, relates to cultures, such as starter cultures, comprising such mutants, and to dairy products fermented with the cultures.
• lactic acid bacteria used intensively in order to bring about the acidification of milk (by fermentation) but also in order to texturize the product into which they are incorporated.
• lactic acid bacteria used in the food industry, there can be mentioned the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium.
• the lactic acid bacteria of the species Streptococcus thermophilus are used extensively alone or in combination with other bacteria such as Lactobacillus delbrueckii subsp. bulgaricus for the production of food products, in particular fermented products. They are used in particular in the formulation of the ferments used for the production of fermented milks, for example yoghurts. Certain of them play a dominant role in the development of the texture of the fermented product. This characteristic is closely linked to the production of polysaccharides.
• novel texturizing strains of lactic acid bacteria in particular of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus, for texturizing food products.
• novel texturizing strains of Lactobacillus delbrueckii subsp bulgaricus which can be used together with novel texturizing strains of Streptococcus thermophilus.
• the inventors of the present invention has previously developed a novel selection method for the identification of improved lactic acid bacteria, such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria, which result in high texture when used for fermenting milk substrate for fermented milk products.
• 2012/052557 relates to that the present inventors have identified a surprisingly relevant link between resistance to D-cycloserine and functionally equivalent antibiotics and improved texturizing properties of the lactic acid bacteria.
• D-cycloserine (D-4-amino-isoxasolidone) is an antibiotic which inhibits alanine racemase, D- alanyl-D-alanine ligase, D-alanyl-alanine synthase and D-alanine permease causing cell lysis.
• D-alanine racemase is essential for the production of D-alanine, an integral part of the peptidoglycan layer of the cell wall.
• Ampicillin is an antibiotic of the beta-lactam class of antibiotics and is effective against many gram-positive bacteria including most lactic acid bacteria. Ampicillin is a competitive inhibitor of enzymes of the DD-transpeptidase type (EC 3.4.16.4). Inhibition of DD-transpeptidase by ampicillin prevents the formation of a peptide bond required for the formation of the bacterial cell wall and ultimately leads to lysis of the cell.
• the inventors of the present invention have surprisingly found that a group of lactic acid bacteria mutants resistant to ampicillin generates high shear stress and/or gel stiffness when the bacteria are used for fermenting milk.
• the present inventors have developed a method for obtaining such texturizing lactic acid bacteria strains which are resistant to the antibiotic ampicillin and/or another antibiotic which inhibits the enzyme DD-transpeptidase.
• the herein described method for obtaining a lactic acid bacteria strain comprises the following two steps: (i) first to screen and select for lactic acid bacteria strains which are resistant to ampicillin, one may term it a resistance to ampicillin that is significantly higher than normally present in natural/wildtype lactic acid bacteria; and
• step (ii) from the pool of ampicillin resistant strains identified in step (i) to screen and select for a lactic acid bacteria strains that has improved texturizing properties.
• Example 2 the present inventors found that from a pool of ampicillin resistant lactic acid bacteria strains it was relatively rapid to screen/select for a lactic acid bacteria strain that has improved texturizing properties. Essentially, the reason for this is that the present inventors have identified that a very high percentage of the ampicillin resistant lactic acid bacteria strain (selected in step (i)) also have improved texturizing properties.
• the first screening and selection for resistance to ampicillin may be seen as a kind of pre-step to rapidly and efficiently be able to screen and select for a lactic acid bacteria strain that has improved texturizing properties.
• a significant advantage of the herein described screening and selection method is that one relatively rapidly and efficiently is able to screen and select for a lactic acid bacteria strain that has improved texturizing properties.
• lactic acid bacteria strain with commercially relevant properties in relation to e.g. low post-acidification when used in preparing fermented milk products
• Example 2 As shown in Example 2 herein approximately 25 % of the first selected ampicillin resistant strains also resulted in significantly higher texture as determined by an efflux time of 28 ml acidified milk from a polystyrene 25-ml pipette of at least 50 seconds. It is submitted that without using the novel screening and selection method as described herein, it would not be possible (or it would take a very long time) to identify a lactic acid bacteria strain that results in such a long efflux time.
• EPS extracellular polysaccharides
• lactic acid bacteria strains are first selected for being resistant to ampicillin, i.e. resistant to ampicillin concentrations that are significantly higher than the ampicillin concentrations tolerated by natural/wildtype lactic acid bacteria.
• the present invention relates to texturizing lactic acid bacteria strains, such as Lactobacillus delbrueckii subsp bulgaricus and
• Streptococcus thermophilus strains which are ampicillin resistant and/or resistant to another antibiotic which inhibits the enzyme DD-transpeptidase and a method for obtaining such strains. Furthermore, the present invention relates to cultures, such as starter cultures, comprising the mutants and to dairy products, such as fermented milk products fermented with the cultures.
• lactic acid bacterium designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid.
• the industrially most useful lactic acid bacteria are found within the order "Lactobacillales” which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp.
• lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria bifidobacteria, i.e. Bifidobacterium spp.
• bifidobacteria i.e. Bifidobacterium spp.
• lactic acid bacteria including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product.
• DVS Direct Vat Set
• Such cultures are in general referred to as "starter cultures” or “starters”.
• milk is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels.
• the milk is cow's milk.
• milk also includes protein/fat solutions made of plant materials, e.g. soy milk.
• milk substrate may be any raw and/or processed milk material that can be subjected to fermentation according to the method of the invention.
• useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream.
• the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder.
• At least part of the protein in the milk substrate is proteins naturally occurring in milk, such as casein or whey protein.
• part of the protein may be proteins which are not naturally occurring in milk.
• the milk substrate Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.
• homogenizing as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.
• “Pasteurizing” as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms.
• pasteurization is attained by maintaining a specified temperature for a specified period of time.
• the specified temperature is usually attained by heating.
• the temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria.
• a rapid cooling step may follow.
• “Fermentation” in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism.
• fermentation in the methods of the invention comprises conversion of lactose to lactic acid.
• Fermentation processes to be used in production of fermented milk products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected so as to support the achievement of the present invention, i.e. to obtain a dairy product in solid or liquid form (fermented milk product).
• a yoghurt starter culture is a bacterial culture which comprises at least one Lactobacillus delbrueckii subsp bulgaricus strain and at least one Streptococcus
• thermophilus strain refers to a fermented milk product obtainable by inoculating and fermenting milk with a composition comprising a Lactobacillus delbrueckii subsp bulgaricus strain and a Streptococcus thermophilus strain.
• texture or “mouthfeel” are meant the product's physical and chemical interaction in the mouth.
• Methods for determining the texture of milk include measuring the shear stress (viscosity), gel stiffness and ropiness of the fermented milk are readily available and known in the art and exemplified herein.
• Viscosity (unit is Pa s) is defined as Shear Stress (Pa) / Shear rate (1/s).
• gel stiffness or “gel firmness” is a measure of how long the structure of a fermented milk product is retained when it is subjected to pressure and is measured in 1 HZ (Pa)
• ropiness refers to the formation of strings and threads and cohesiveness in the fermented milk product. Ropiness is defined and measured as described in Int. Dairy J. 16(2); 111-118 (Folkenberg et al. 2006).
• resistant to ampicillin herein means that a particular mutated bacterial strain is not killed, or killed significantly more slowly compared to the corresponding non-mutated strain from which the mutated strain is derived in the presence of said antibiotic in the culture medium. Dependent on the concentration of the antibiotic compound in the culture medium, resistance can also be reflected by altered growth properties of the mutated and non-mutated strains.
• Non-mutated strains which can be used as sensitive reference strains in the assessment of resistance preferably include the strain CHCC13995.
• a lactic acid bacteria strain which is resistant to the antibiotic ampicillin is herein defined as a lactic acid bacteria strain, wherein the amount of the antibiotic ampicillin that reduces the OD(5oo measured growth, after 20 hours growth at 37°C, with 20% in a medium suitable for the growth of the lactic acid bacteria strain (e.g. MRS medium for Lactobacillus delbrueckii subsp. bulgaricus strains and M17 containing 2% (w/v) lactose for Streptococcus thermophilus) as compared to the OD600 measured growth in the medium without the antibiotic ampicillin is higher than 400 ng/ml.
• a mutant lactic acid bacteria strain which is resistant to ampicillin is herein further defined by that the minimum inhibitory concentration (MIC) value read in an E-test is at least 1 increment higher for the mutant lactic acid bacteria strain than for the mother strain from where the mutant strain is derived.
• MIC minimum inhibitory concentration
• mutant should be understood as a strain derived, or a strain which can be derived from a strain of the invention (or the mother strain) by means of e.g. genetic engineering, radiation and/or chemical treatment.
• the mutant can also be a spontaneously occurring mutant. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding viscosity, gel stiffness and ropiness) as the mother strain. Such a mutant is a part of the present invention.
• mutant refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl- N'-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring mutant.
• a mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out.
• variant should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties e.g. regarding viscosity, gel stiffness and ropiness).
• Such variants which may be identified using appropriate screening techniques, are a part of the present invention.
• antibiotics with the same mode of action or the same targets as ampicillin can be used alone or in combination with ampicillin to isolate mutants of the type described herein.
• the present invention also encompasses the use of such other functionally equivalent antibiotics, such as other inhibitors of DD- transpeptidase.
• antibiotics include, but are not limited to, amoxicillin, penicillin G, procaine penicillin, benzathine penicillin, and penicillin V.
• the present invention relates to a lactic acid bacteria strain characterized by that:
• the lactic acid bacteria strain is resistant to the antibiotic ampicillin and/or another antibiotic which inhibits the enzyme DD-transpeptidase, defined by that the amount of the antibiotic that reduces the OD 50 o measured growth, after 20 hours growth at 37°C, with 20% in a medium suitable for growth of the lactic acid bacteria strain as compared to the OD 50 o measured growth in the medium without the antibiotic is higher than 400 ng/ml; and
• the efflux time from a polystyrene 25-ml pipette of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the lactic acid bacteria strain and acidified at 37°C for 20 hours is at least 50 seconds.
• the antibiotic of step (i) is ampicillin.
• Both the assay for determining the amount of antibiotic which reduces the OD 50 o measured growth by 20% of point (i) and the assay for determining the efflux time of 28 ml acidified milk from a polystyrene 25-ml pipette of point (ii) are based on known, commercially available standard elements.
• the lactic acid bacteria strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. In a more preferred embodiment the lactic acid bacteria strain is a Lactobacillus delbrueckii subsp.
• growth with 20% of point (i) is higher than 500 ng/ml, such as higher than 600 ng/ml, such as higher than 700 ng/ml.
• the efflux time of point (ii) is at least 60 seconds, such as at 15 least 70 seconds, such as at least 80 seconds, such as at least 90 seconds, such as at least 100 seconds, such as at least 110 seconds.
• the present invention relates to a lactic acid bacteria strain characterized by that:
• the lactic acid bacteria strain is resistant to the antibiotic ampicillin and/or another
• MIC Inhibitory Concentration
• the antibiotic of step (i) is ampicillin.
• the lactic acid bacteria strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. In a more preferred embodiment the lactic acid bacteria strain is a Lactobacillus delbrueckii subsp. bulgaricus strain.
• the MIC for the mutant lactic acid bacteria strain of point (i) is at least 2 increments, such as at least 3 increments, such as at least 4 increments, such as at least 5 increments, higher in an E-test than the MIC for the mother strain.
• the efflux time of point (ii) is at least 50 seconds, such as at least 60 seconds, such as at least 70 seconds, such as at least 80 seconds, such as at least 90 seconds, such as at least 100 seconds, such as at least 110 seconds.
• a third aspect of the present invention is directed to a lactic acid bacteria strain selected from the group consisting of the Lactobacillus delbrueckii subsp. bulgaricus strain CHCC15466 that was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 25852 and mutants and variants derived thereof.
• mutants derived thereof relates to mutant strains obtained by using the deposited strain as starting material and wherein the mutants retain the essential property of the deposited strain, wherein said essential property is that the efflux time of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the lactic acid bacteria strain and acidified at 37°C for 20 hours, from a polystyrene 25-ml pipette is at least 50 seconds.
• a fourth aspect of the present invention relates to a composition
• a composition comprising from 10 4 to 10 14 CFU/g of a lactic acid bacteria strain according to any of claims 1 to 6.
• the composition comprises at least one Lactobacillus delbrueckii subsp. bulgaricus strain.
• the composition comprises at least one Lactobacillus delbrueckii subsp. bulgaricus strain and at least one Streptococcus thermophilus strain.
• composition is usable as a starter culture.
• composition is in frozen, freeze-dried or liquid form.
• the lactic acid bacteria strain (which is resistant to ampicillin and/or another antibiotic which inhibits DD-transpeptidase and which has improved texturizing properties) according to the present invention may preferably be used for preparing a fermented milk product.
• the dose and administration may be done according to the art.
• a fifth aspect of the present invention relates to a method for preparing a fermented milk product, comprising fermenting a milk substrate with the lactic acid bacteria strain according to the first, second or third aspect of the invention or the composition according to the fourth aspect of the present invention.
• a sixth aspect relates to a fermented milk product obtainable by the method according to the fourth aspect of the invention.
• a seventh aspect relates to a fermented milk product comprising the lactic acid bacteria strain according to the first, second or third aspect of the invention or the composition according to the fourth aspect of the invention.
• An eighth aspect of the present invention is directed to use of the lactic acid bacteria strain according to the first, second or third aspect of the invention or the composition according to the fourth aspect of the invention for the preparation of a dairy product.
• the dairy product is a fermented milk product.
• the fermented milk product is a yoghurt.
• a ninth aspect of the present invention relates to a method for obtaining a lactic acid bacteria strain, said method comprising :
• the method comprises:
• step b) selecting and isolating, from the pool of lactic acid bacteria strains which are resistant to the antibiotic ampicillin of step a), a lactic acid bacteria strain wherein the
• 15 efflux time of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the lactic acid bacteria strain and acidified at 37°C for 20 hours from a polystyrene 25-ml pipette is at least 50 seconds.
• the lactic acid bacteria strain is selected from the group consisting 20 of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.
• the lactic acid bacteria strain is a Lactobacillus delbrueckii subsp.
• the amount of the antibiotic that reduces the OD 50 o measured 25 growth with 20% of point (i) is higher than 500 ng/ml, such as higher than 600 ng/ml, such as higher than 700 ng/ml.
• the efflux time of point (ii) is at least 60 seconds, such as at least 70 seconds, such as at least 80 seconds, such as at least 90 seconds, such as at least 30 100 seconds, such as at least 110 seconds.
• a tenth aspect is directed to a method for obtaining a lactic acid bacteria strain, said method comprising :
• the mutant lactic acid bacteria strains resistant to the antibiotic ampicillin and/or to another antibiotic which inhibits the enzyme DD-transpeptidase, defined by that the Minimum Inhibitory Concentration (MIC) for the mutant lactic acid bacteria strains is at least 1 increment higher in an E- test than the MIC for the mother strain from which the mutant lactic acid bacteria
• 40 strains are derived; and c) selecting and isolating, from the pool of mutant lactic acid bacteria strains which are resistant to ampicillin and/or to another antibiotic which inhibits the enzyme DD- transpeptidase of step b), a mutant lactic acid bacteria strain wherein the efflux time from a polystyrene 25-ml pipette of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the mutant lactic acid bacteria strain and acidified at 37°C for 20 hours is at least double the time as the efflux time from a polystyrene 25-ml pipette of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the mother strain and acidified at 37°C for 20 hours.
• the method comprises:
• Concentration (MIC) for the mutant lactic acid bacteria strains is at least 1 increment higher in an E-test than the MIC for the mother strain from which the mutant lactic acid bacteria strains are derived;
• step b) selecting and isolating, from the pool of mutant lactic acid bacteria strains which are resistant to ampicillin of step b), a mutant lactic acid bacteria strain wherein the efflux time from a polystyrene 25-ml pipette of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the mutant lactic acid bacteria strain and acidified at 37°C for 20 hours is at least double the time as the efflux time from a polystyrene 25-ml pipette of 28 ml of full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the mother strain and acidified at 37°C for 20 hours.
• the lactic acid bacteria mother strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.
• the lactic acid bacteria strain is a Lactobacillus delbrueckii subsp. bulgaricus strain.
• the mother strain is the Lactobacillus delbrueckii subsp. bulgaricus strain CHCC13995 that was deposited with
• the MIC for the mutant lactic acid bacteria strain of point (i) is at least 2 increments, such as at least 3 increments, such as at least 4 increments, such as at least 5 increments, higher in an E-test than the MIC for the mother strain.
• the efflux time of point (ii) from a polystyrene 25-ml pipette of 28 ml full fat cow milk containing 2% (w/v) skimmed milk powder, inoculated with at least 10 4 CFU/ml of the mutant lactic acid bacteria strain and acidified at 37°C for 20 hours is at least 50 seconds, such as at least 60 seconds, such as at least 70 seconds, such as at least 80 seconds, such as at least 90 seconds, such as at least 100 seconds, such as at least 110 seconds.
• the present invention relates to a lactic acid bacteria strain obtainable by a method according to the eighth or ninth aspect of the invention.
• suitable media include the known M 17 agar medium having the following composition : agar, 12.75 g/L
• soyapeptone papainic
• yeast extract 2.5 g/L and M 17 broth medium with this composition : ascorbic acid, 0.5 g/L
• suitable media include the known MRS medium.
• MRS agar medium has the following composition : Bacto Proteose Peptone no.3 10 g/l
• Bacto Agar 15 g/l and MRS broth medium has this composition :
• the M 17 medium is a medium that is considered to be suitable for growth of Streptococcus thermophilus and the MRS medium is a medium that is considered to be suitable for growth of Lactobacillus delbrueckii subsp. bulgaricus.
• the specific M 17 and MRS media concentrate may be supplied from different suppliers and independently of the specific supplier one will (within standard measurement uncertainty) get the same herein relevant result of ampicillin resistance for a herein relevant strain of interest.
• the method is illustrated using a strain of Lactobacillus delbrueckii subsp. bulgaricus.
• M17 medium with lactose added to 2 % w/v should be used instead of MRS medium.
• a Lactobacillus delbrueckii subsp. bulgaricus strain of interest is inoculated into 10 ml MRS broth medium and grown for at least 20 hours at 37°C under anaerobic conditions.
• a cotton swab is dipped in the culture and used to streak out on the entire surface of a MRS agar plate (90 mm in diameter).
• An ampicillin E-test stick (0.016-256 ⁇ g/ml) for Antimicrobial
• Susceptibility Testing (Biomerieux, cat. #501558) is placed on top of the agar, and the plate is incubated for not more than 24 hours at 37°C under anaerobic conditions.
• the minimum inhibitory concentration (MIC) of ampicillin is the lowest concentration that will inhibit the visible growth of the plated strain and is read at the point where the elliptical zone of inhibition intersects the E-test strip.
• a Lactobacillus delbrueckii subsp. bulgaricus cell that has an increased resistance to ampicillin as discussed herein, is herein defined as a Lactobacillus delbrueckii subsp. bulgaricus cell, where the MIC value read is at least 1 increment higher as noted on the E-strip than for the mother strain.
• Cells that are capable of complying with this increased resistance to ampicillin criteria are herein defined as cells that are resistant to ampicillin in the ampicillin resistance assay of this Example 1.
• EXAMPLE 2 Use of ampicillin to isolate mutants of Lactobacillus delbrueckii subsp. bulgaricus with improved rheological properties
• Lactobacillus delbrueckii subsp. bulgaricus CHCC15466 ampicillin resistant mutant of
• bulgaricus strain CHCC13995 cells derived from the growth of a single colony were inoculated anaerobicaiiy into 10 ml MRS broth containing one of the following amounts of ampicillin, 0 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml or 700 ng/ml, and grown for at least 20 hours at 37°C.
• the concentration of ampicillin that reduces the OD 50 o measured anaerobic growth of Lactobacillus delbrueckii subsp. bulgaricus with at least 20 % as compared to the growth in MRS medium without ampicillin (i.e. with 0 ng/ml ampicillin) is higher than 300 ng/ml.
• a rheological screening analysis was done by using a simple pipette viscosity test.
• the ampicillin resistant mutants were pre-tested for their viscosity (before real rheology testing) by measuring the efflux time from a polystyrene 25-ml pipette (CELLSTAR®) loaded with 28 ml of acidified milk:
• the acidified milk was gently homogenized by stirring before loading into a polystyrene 25-ml pipette to the top (28 ml) and for each sample the efflux time from the pipette was measured 3 times.
• Table 1 lists the result of the pipette viscosity test on milk fermented at 37°C for 20 hours by 21 ampicillin resistant mutants. The results show that most of these mutants result in a higher viscosity than mother strain. 6 out of the 21 ampicillin resistant mutants result in an efflux time which is double that of the mother strain and approximately 25% of the ampicillin resistant mutants result in an efflux time of at least 50 seconds.
• This mutant derivative was 5 designated CHCC15466 and was used for rheological test described in Example 3.
• Lactobacillus delbrueckii subsp. bulgaricus ampicillin resistant mutants described herein may be incorporated into a culture, such as a starter culture, which produces a desirable high 15 level of texture.
• EXAMPLE 3 Use of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus ampicillin resistant mutants for preparation of a fermented milk product.
• the yoghurt cultures were mixed at a ratio of 9: 1 of Streptococcus thermophilus : Lactobacillus delbrueckii subsp bulgaricus and inoculated into full fat cow milk with 2% added skimmed milk powder and fermented at 40°C to a final pH of 4.50.
• the following mixtures were analyzed after 5 days storage at 4°C using the Stresstech rheometer. Ropiness is measured as described by Folkenberg et al. (2006. Int. Dairy J. 16(2); 111-118)
• Lactobacillus delbrueckii subsp. bulgaricus ampicillin resistant mutant described herein may be incorporated into a culture, such as a starter culture, which produces a desirable high level of texture in a fermented milk product such as yoghurt.
• the strain of Streptococcus thermophilus CHCC4895 was deposited with Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 29 March 2007 under the accession number DSM 19242.
• the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC13995 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 22 September 2010 under the accession number DSM 24021.
• the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC15466 was deposited with
• the Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.

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